Novel primate chitinases and uses therefor

Abstract

Novel primate chitinases (pNC) and nucleic acids encoding these chitinases are disclosed. The pNCs of the invention are homologous to human acidic mammalian chitinases (hAMCase) and human chitotriosidase, in terms of their exon/intron genomic structure and conserved catalytic sites. The polypeptides and nucleic acids of the invention can be used to develop, and/or screen for, modulators of chitinase-activities, e.g., antisense, antibodies, RNAi. The modulators identified using methods and reagents disclosed herein can be used to reduce chitinase-associated inflammatory conditions.

1 . An isolated nucleic acid molecule comprising a nucleotide sequence selected from the group consisting of:
 a) a nucleotide sequence that encodes a pNC polypeptide comprising a chitinase catalytic domain from about amino acid 22 to about amino acid 408 of SEQ ID NO:2, a linker sequence from about amino acid 409 to about amino acid 427 of SEQ ID NO:2, and a chitin-binding domain from about amino acid 428 to about amino acid 474 of SEQ ID NO:2;   b) a nucleotide sequence that encodes a polypeptide comprising the amino acid sequence of SEQ ID NO:2;   c) a nucleotide sequence that encodes a polypeptide comprising an amino acid sequence at least 83% identical to the amino acid sequence of SEQ ID NO:2;   d) a nucleotide sequence comprising the sequence of SEQ ID NO:1;   e) a nucleotide sequence comprising a nucleotide sequence at least 85%, identical to the nucleotide sequence of SEQ ID NO:1;   f) a nucleotide sequence comprising a fragment of at least 100 contiguous nucleotides of SEQ ID NO:1;   g) a nucleotide sequence which hybridizes under highly stringent hybridization conditions to a nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO:1, or a complement thereof; and   h) a nucleotide sequence which comprises nucleotides 513-524, 780-784, 830-835, 843-847, 1117-1124, 1211-1237, 1211-1226, 1220-1237, 1262-1288, 1295-1305, 1312-1320, 1360-1376, 1404-1430, 1404-1414 and 1418-1430, of SEQ ID NO:1.   
     
     
         2 . The nucleic acid molecule of  claim 1  further comprising vector nucleic acid sequences. 
     
     
         3 . The nucleic acid molecule of  claim 1  further comprising nucleic acid sequences encoding a heterologous polypeptide. 
     
     
         4 . A host cell which contains the nucleic acid molecule of  claim 1 . 
     
     
         5 . The host cell of  claim 4  which is a mammalian host cell. 
     
     
         6 . A non-human mammalian host cell containing the nucleic acid molecule of  claim 1 . 
     
     
         7 . An isolated polypeptide selected from the group consisting of:
 a) a pNC polypeptide comprising a chitinase catalytic domain from about amino acid22 to about amino acid 408 of SEQ ID NO:2, a linker sequence from about amino acid409 to about amino acid 427 of SEQ ID NO:2, and a chitin-binding domain from about amino acid428 to about amino acid 474 of SEQ ID NO:2;   b) a polypeptide comprising the amino acid sequence of SEQ ID NO:2;   c) a polypeptide comprising an amino acid sequence at least 83% identical to the amino acid sequence of SEQ ID NO:2;   d) a polypeptide comprising an amino acid sequence encoded by a nucleotide sequence comprising the sequence of SEQ ID NO:1;   e) a polypeptide comprising an amino acid sequence encoded by a nucleotide sequence comprising a nucleotide sequence at least 85% identical to the nucleotide sequence of SEQ ID NO:1;   f) a polypeptide comprising an amino acid sequence encoded by a nucleotide sequence comprising a fragment of at least 100 contiguous nucleotides of SEQ ID NO:1;   g) a polypeptide comprising an amino acid sequence encoded by a nucleotide sequence which hybridizes under highly stringent hybridization conditions to a nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO:1, or a complement thereof;   h) a fragment of a pNC polypeptide of at least 90 contiguous amino acids of SEQ ID NO:2; and   i) a fragment of a pNC polypeptide comprising about amino acids 22-408, 409-427, 428-474, or about amino acids 1-21, 22-120, 22-180, 22-240, 22-300, 22-360, 22-400, 120-150, 150-170, 170-180, 180-190, 190-220, 220-250, 250-270, 270-280, 280-290, 290-300, 300-320, 320-330, 330-340, 340-350, 350-360, 360-390, 390-400, 400-410, 410-420, 420-430, 430-440, 440-450, 450-460, 460-470, or 470-476, of SEQ ID NO:2.   
     
     
         8 . The polypeptide of  claim 7  further comprising heterologous amino acid sequences. 
     
     
         9 . A method for producing a pNC polypeptide, comprising culturing the host cell of  claim 4  under conditions in which the nucleic acid molecule is expressed. 
     
     
         10 . An antibody molecule which selectively binds to a polypeptide of  claim 7 , wherein said antibody molecule binds to human AMCase with an affinity that is less than 30% compared to the affinity of the antibody molecule to the pNC. 
     
     
         11 . An antibody molecule with binds to a polypeptide of  claim 7  and human AMCase with substantially equal affinities. 
     
     
         12 . The antibody molecule of  claim 10  or  11 , which can reduce one or more activities of a pNC polypeptide. 
     
     
         13 . A method of providing an antibody molecule that specifically binds to a pNC polypeptide, comprising:
 (i) providing a non-human chitinase, or fragment thereof, wherein said non-human chitinase is at least 85% identical to a corresponding portion of a human chitinase protein;   (ii) obtaining an antibody molecule that specifically binds to the non-human chitinase or fragment thereof; and   (iii) evaluating if the antibody molecule specifically binds to the human chitinase protein.   
     
     
         14 . The method of  claim 13 , further comprising administering the antibody molecule to a subject. 
     
     
         15 . The method of  claim 13 , wherein the subject is a human or non-human primate. 
     
     
         16 . A method of evaluating the ability of a test compound to interact with a pNC polypeptide or nucleic acid, comprising:
 contacting a pNC polypeptide or nucleic acid with the test compound; and   evaluating a change in one or more activities of the pNC polypeptide or nucleic acid in the presence of the test compound, relative to a control sample without the test compound.   
     
     
         17 . The method of  claim 16 , wherein the activities evaluated comprise a change in the expression, binding, or enzymatic activity of the pNC polypeptide or nucleic acid. 
     
     
         18 . The method of  claim 16 , wherein the contacting step is effected in vitro or in vivo. 
     
     
         19 . A method for identifying a test compound, which inhibits the activity of a pNC polypeptide, or the expression of a pNC nucleic acid, comprising: contacting the pNC polypeptide or nucleic acid with a test compound; and determining the effect of the test compound on the activity of the polypeptide or nucleic acid to thereby identify a compound which inhibits the activity of the polypeptide or nucleic acid. 
     
     
         20 . The method of  claim 19 , wherein the test compound is a peptide, a phosphopeptide, a small molecule, a nucleic acid, an antisense molecule, a ribozyme, an RNAi, a triple helix molecule, an antibody molecule, a chitinase inhibitor or an analogue thereof, or any combination thereof. 
     
     
         21 . The method of  claim 19 , wherein the test compound is an inhibitor of chitinase activity. 
     
     
         22 . The method of  claim 19 , wherein the contacting step is effected by administering the test compound to a subject, and evaluating a change in one or more symptoms of a chitinase-associated disorder. 
     
     
         23 . The method of  claim 22 , wherein the symptoms evaluated comprise one or more of: (i) detecting a change in the number of eosinophils, macrophages, or neutrophils into the airways; (ii) measuring eotaxin levels; (iii) detecting in basophil histamine release; or (iv) detecting IgE titers, wherein a reduction, in the level of one or more of (i)-(iv) comparing before and after treatment) indicates that the test compound is effectively reducing airway eosinophilia in the subject. 
     
     
         24 . An inhibitor of chitinase activity identified by the method of  claim 19 .